Abstract 5338: A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy

Abstract

Abstract It has been well-established that hepatic drug metabolism may have significant impact on the efficacy and off-target toxicity of anticancer drugs. We present here an in vitro assay, the metabolism-dependent cytotoxicity assay (MDCA), for the evaluation of the roles of specific pathways on the efficacy of anticancer drugs. The assay employs a novel in vitro hepatic metabolic system, the permeabilized cofactor-supplemented human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system for the evaluation of the in vitro cytotoxicity of anticancer agents towards a designated tumor and nontumor cells. MMHH are derived from cryopreserved human hepatocytes isolated from human livers procured for but not used for transplantation provided to our laboratory from the International Institute for the Advancement of Medicine. We have previously demonstrated that MMHH retained all drug metabolizing enzyme pathways present in human hepatocytes. The advantages of MMHH include the following: 1. Drug metabolizing enzyme activities are not affected by cytotoxicity of the drug substrate, a major limitation of intact human hepatocytes; and 2. Drug metabolism pathways can be selected by cofactor specification (e.g. NADPH/NAD+ for phase 1 oxidation; Uridine 5′-diphosphoglucuronic acid (UDPGA) for glucuronidation; 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for sulfation; N-acetyl coenzyme A for N-acetylation). We present here a proof-of-concept study to evaluate the role of various hepatic metabolic pathways towards the cytotoxicity of cyclophosphamide, a commonly used drug for the treatment of renal carcinoma, towards a renal cancer cell line, HEK293 cells. We observed that NADPH enhanced the cytotoxicity, consistent with the known metabolic activation of cyclophosphamide by P450 isoforms (e.g. CYP2B6; CYP3A4) to the known metabolic activation of cyclophosphamide to 4-hydroxycloophosphamide, followed by the formation of the ultimate cytotoxic metabolites phosphoramide mustard and acrolein, which are responsible for its anticancer properties. In addition, we found that the addition of L-glutathione attenuated cyclophosphamide cytotoxicity towards the HEK293 cells, an observation consistent with the roles of this cellular detoxification system in the resistance of tumor cells to chemotherapeutic agents. Minimal effects were observed for UDPGA and PAPS, consistent with the known noninvolvement of these phase 2 conjugating pathways in cyclophosphamide metabolism. MDCA represents an in vitro experimental system that can be routinely applied towards the discovery and development of novel anticancer drugs to evaluate the roles of drug metabolism in efficacy and safety, with various cancer cells (e.g. prostate carcinoma, neuroblastoma) and cells from normal tissues (e.g. cardiomyocytes, intestinal mucosal cells), respectively, as target cells. Citation Format: Albert P. Li, Hong Wei. A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5338.